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Synthetic biology is a growing field at the interface of the engineering and biological sciences which aims to apply rational engineering design principles in biological contexts. It has many applications ranging from healthcare and the environment to industrial chemical synthesis and computation. When researchers attempt to apply synthetic biological tools to tackle these problems, they often follow a similar design-build-test cycle to that employed in traditional engineering disciplines. However, there are major challenges at each step in this cycle due to the variability inherent in biological systems, and our limited ability to understand and measure their behaviour. This thesis addresses select problems in each step of the design-build-test cycle as applied to synthetic biology, from a control-theoretic perspective. To aid the design process of synthetic biological circuits I begin by using traditional control-theoretic analysis to derive structural requirements that biological systems must satisfy if they are to achieve behaviours such as disturbance rejection. I demonstrate how these requirements can be used to inform the biological design process, and highlight the challenges that must be overcome for effective noise attenuation in such circuits. To refine the build/creation process for synthetic biological systems, I describe the experimental implementation and subsequent analysis of new synthetic biological feedback controllers built from two classes of components; short RNA and Recombinase proteins. Our results demonstrate how these circuits can be used to tune (or create) feedback loops, and reduce the influence of cellular noise on the performance of our synthetic circuits. This work thus has the double benefit of demonstrating new approaches to the engineering of biological systems, as well as creating well-characterised biological circuits that can be applied directly to a range of applications. Finally, to improve the testing/characterisation process for synthetic biological systems I develop a novel biological approach to measuring cell-to-cell variability, as well as a new automated experimental platform: The Turbidostat. This device provides many of the important measurement and actuation capabilities required for synthetic biological research in a cheap and easy-to-use robotic platform. It can thus be used to automate a diverse range of characterisation experiments that would otherwise be highly laborious and prone to variability/noise. I perform several validation experiments, demonstrating this device’s broad range of capabilities.

We evaluated immune biomarker profiles in HIV-infected adults (n=398) from 5 African countries. Although all biomarkers decreased after ART initiation, CXCL10, LBP, CRP, sCD163 and sCD14 were significantly higher during ART than in an HIV-negative reference group (n=90), indicating persistent monocyte/macrophage activation, inflammation and microbial translocation. Before ART initiation, high HIV viral load was associated with elevated CXCL10 and tuberculosis coinfection was associated with elevated sCD14. High pre-ART levels of each biomarker strongly predicted residual immune activation during ART. CCL2, LBP, CRP, IL-6 were differentially expressed between countries. Further research is needed on the clinical implications of residual immune dysregulation.

Accurate control of a biological process is essential for many critical functions in biology, from the cell cycle to proteome regulation. To achieve this, negative feedback is frequently employed to provide a highly robust and reliable output. Feedback is found throughout biology and technology, but due to challenges posed by its implementation, it is yet to be widely adopted in synthetic biology. In this paper we design a synthetic feedback network using a class of recombinase proteins called integrases, which can be re-engineered to flip the orientation of DNA segments in a digital manner. This system is highly orthogonal, and demonstrates a strong capability for regulating and reducing the expression variability of genes being transcribed under its control. An excisionase protein provides the negative feedback signal to close the loop in this system, by flipping DNA segments in the reverse direction. Our integrase/excisionase negative feedback system thus provides a modular architecture that can be tuned to suit applications throughout synthetic biology and biomanufacturing that require a highly robust and orthogonally controlled output.

Systemic immune activation has emerged as an essential component of the immunopathogenesis of HIV. It not only leads to faster disease progression, but also to accelerated decline of overall immune competence. HIV-associated immune activation is characterized by an increase in proinflammatory mediators, dysfunctional T regulatory cells, and a pattern of T-cell-senescent phenotypes similar to those seen in the elderly. These changes predispose HIV-infected persons to comorbid conditions that have been linked to immunosenescence and inflamm-ageing, such as atherosclerosis and cardiovascular disease, neurodegeneration, and cancer. In the antiretroviral treatment era, development of such non-AIDS-defining, age-related comorbidities is a major cause of morbidity and mortality. Treatment strategies aimed at curtailing persistent immune activation and inflammation may help prevent the development of these conditions. At present, the most effective strategy appears to be early antiretroviral treatment initiation. No other treatment interventions have been found effective in large-scale clinical trials, and no adjunctive treatment is currently recommended in international HIV treatment guidelines. This article reviews the role of systemic immune activation in the immunopathogenesis of HIV infection, its causes and the clinical implications linked to immunosenescence in adults, and the therapeutic interventions that have been investigated.

Cysteinyl leukotrienes (cysLTs) are produced predominantly by cells of the innate immune system, especially basophils, eosinophils, mast cells, and monocytes/macrophages. Notwithstanding potent bronchoconstrictor activity, cysLTs are also proinflammatory consequent to their autocrine and paracrine interactions with G-protein-coupled receptors expressed not only on the aforementioned cell types, but also on Th2 lymphocytes, as well as structural cells, and to a lesser extent neutrophils and CD8+ cells. Recognition of the involvement of cysLTs in the immunopathogenesis of various types of acute and chronic inflammatory disorders, especially bronchial asthma, prompted the development of selective cysLT receptor-1 (cysLTR1) antagonists, specifically montelukast, pranlukast, and zafirlukast. More recently these agents have also been reported to possess secondary anti-inflammatory activities, distinct from cysLTR1 antagonism, which appear to be particularly effective in targeting neutrophils and monocytes/macrophages. Underlying mechanisms include interference with cyclic nucleotide phosphodiesterases, 5′-lipoxygenase, and the proinflammatory transcription factor, nuclear factor kappa B. These and other secondary anti-inflammatory mechanisms of the commonly used cysLTR1 antagonists are the major focus of the current review, which also includes a comparison of the anti-inflammatory effects of montelukast, pranlukast, and zafirlukast on human neutrophils in vitro, as well as an overview of both the current clinical applications of these agents and potential future applications based on preclinical and early clinical studies.

Viral drug susceptibility is associated with virologic response to new treatments. Standardized drug resistance tests are now available, and data from some clinical trials suggest that the use of drug resistance testing may be associated with improved virologic outcome. However, drug resistance testing is complex in terms of performance, interpretation and clinical application. HIV-1 drug resistance testing is used across Europe in patient management, but not in a consistent manner. This is due to differences in the national approaches to treatment, treatment management and reimbursement, as well as availability of tests. National guidelines only exist in some countries. In addition, the laboratory quality assurance and quality control standards are not applied uniformly. The EuroGuidelines Group was established to formulate clinical as well as laboratory guidelines for the use of HIV-1 drug resistance testing that are specific for the European setting. The group is comprised of academic clinicians and virologists, scientist from the industry and representatives of the patient community. The panel of experts will review these guidelines and update them on a yearly basis as new scientific evidence becomes available. (C) 2001 Lippincott Williams & Wilkins.